Beef insulin preparations

ABSTRACT

A STABLE AND PHARMACEUTICALLY ACCEPTABLE SOLUTION OF INSULIN, COMPRISING BEEF INSULIN WHICH HAS A MOLECULAR WEIGHT OF 5734, SUBSTANTIALLY THE FOLLOWING ELEMENTARY ANALYSIS: CARBON 53.21%, HYDROGEN 6.63%, NITROGEN 15.88%, SULPHUR 3.36%, AND OXYGEN 20.92% (BY DIFFERENCE), AND CHARACTERISTICALLY ALANINE, SERINE, AND VALINE IN THE 8TH, 9TH AND 10TH POSITION OF THE A CHAIN AND ALANINE IN THE 30TH POSITION OF THE B CHAIN, AND HAS (1) A PROTAMINASE CONTENT CAUSING LESS THAN ABOUT 15% W./W. LOSS AT 37*C. IN 30 DAYS WHEN THE INSULIN IS CONVERTED INTO A PROTAMINE-CONTAINING ISOPHANE INSULIN SUSPENSION, (2) AN OPTICAL DENSITY (ABSORBENCE), CORRECTED TO A MANIFOLD DILUTED SAMPLE, OR LESS THAN ABOUT 0.5 AS A 1% ACID SOLUTION IN A 4 CM. CELL AT A WAVELENGTH OF 396 MU, (3) AN OPTICAL DENSITY (ABSORBENCE) OF LESS THAN ABOUT 0.03 AT A WAVELENGTH OF 400 MU WHEN MEASURED AS A 40 INTERNATIONAL UNITS/ML. SOLUTION IN A 1 CM. CELL AT A PH OF 7.4 AFTER STANDING AT 5*C. FOR EIGHT DAYS, WITH AN INSTRUMENT ADAPTED TO EXCLUDE SCATTERED LIGHT, AND, (4) AN INFRA-RED SPECTRUM SUCH AS SHOWN IN THE DRAWING; AS AN AQUEOUS SOLUTION, ADJUSTED TO A PH BETWEEN PH7 AND 8.

April 18, 1972 G. A. STEWART 3,657,425

BEEF INSULIN PREPARATIONS Filed March l2, 1969 Dia@ WQ MME United States Patent O 3,657,425 BEEF INSULIN PREPARATIONS George Alexander Stewart, London, England, assignor to Burroughs Wellcome Co.

Continuation-impart of applications Ser. No. 458,769,

May 25, 1965, and Ser. No. 668,415, Sept. 18, 1967.

This application Mar. 12, 1969, Ser. No. 806,681

Int. Cl. A61k I7/ 02 U.S. Cl. 424-178 3 Claims ABSTRACT OF THE DISCLOSURE A stable and pharmaceutically acceptable solution of insulin, comprising beef insulin which has a molecular weight of 5734, substantially the following elementary analysis: carbon 53.21%, hydrogen 6.63%, nitrogen 15.88%, sulphur 3.36%, and oxygen 20.92% (by difference), and characteristically alanine, serine, and valine in the 8th, 9th and 10th position of the A chain and alanine in the 30th position of the B chain, and has (1) a protaminase content causing less than about 15% w./w. loss at 37 C. in 30 days when the insulin is converted into a protamine-containing isophane insulin suspension,

(2) an optical density (absorbence), corrected to a manifold diluted sample, of less than about 0.5 as a 1% acid solution in a 4 cm. cell at a wavelength of 396 ma,

(3) an optical density (absorbence) of less than about 0.03 at a wavelength of 400 ma when measured as a 40 international units/ml. solution in a l cm. cell at a pH of 7.4, after standing at 5 C. for eight days, with an instrument adapted to exclude scattered light, and,

(4) an infra-red spectrum such as shown in the drawing; as an aqueous solution, adjusted to a pH between pH 7 and 8.

The present invention relates to insulin preparations. This application is a continuation-in-part of earlier applications, Ser. Nos. 458,769, filed on May 25, 1965, and 668,415, tiled on September 18, 1967, both now abandoned.

In addition to the clinically useful and important prolonged acting insulin preparations, there has been a demand for a quick acting type of insulin, which is inexpensive and stable, and is also compatible with the neutral prolonged acting preparations usually containing suspended insulin in an insoluble or slightly soluble form. There is a method described in the specification of British Pat. No. 840,870, which provides a quick acting neutral insulin preparation, but suffers from the disadvantage that it relies on pig insulin, which is less readily available than beef insulin, and is therefore more expensive.

It has also been suggested (Nature, 1966, 212, 766) that since beef insulin considerably differs from human or pig insulin, the serum of patients regularly treated with beef insulin preparations contains antibodies against such insulin which neutralise a proportion of the dose. A risk arises, however, if such a patient is unknowingly administered pig insulin, as such insulin may suddenly produce a larger hypoglycaemic effect, which could be fatal in certain situations, as when driving.

An object of the present invention is to provide a quick acting insulin preparation which has not only the desired properties of inexpensiveness, stability and is compatible with neutral prolonged acting preparations, but can also be administered without risk to patients previously treated with the usual types of beef insulin, and is less likely to cause undesirable reactions at the site of injection because of a physiological pH of the injected medium, than are the commonly used acid insulin solutions.

Beef insulin, like other mammalian insulins, is a polypeptide which contains 51 amino acids arranged in two interlinked chains A and B (cf. Brown et al., Biochem. J., 1955, y6.0, 556). Beef insulin has a molecular weight of M=5734 and differs from other known mammalian and human insulins in that it characteristically has alanine,

I serine and valine in the 8th, 9th and 10th positions of the A chain and alanine in the 30th position of the B chain. Thus it is possible for us to determine the origin of a sample of insulin by analysing its chemical composition. The elemental analysis of beef insulin, corresponding to the known composition, is (on theory) as follows (percent): carbon, 53.21; hydrogen, 6.63; nitrogen, 15.88; sulfur, 3.36 and oxygen, 20.92 (by difference).

It has now been found that the failure to obtain sufficiently stable and reliable neutral solutions of beef insulin has hitherto been due to the fact that small amounts of enzymes or other, so called non-insulin proteins which frequently contaminate the usual qualities of crystalline beef insulin, have caused opalescence, decomposition or loss of biological activity in solution on prolonged standing. Furthermore, according to this discovery, crystalline beef insulin which has been purified so as to contain only negligible amounts of these enzymes and proteins is suitable to form a stable insulin solution in an aqueous medium at a pH between pH 7 and 8. Stability in this context lmeans that the insulin solution remains clear and bright, does not show any significant loss of biological activity and is pharmaceutically acceptable after a storage of at least three months and preferably six months at 5 C. or 25 C., and therefore possesses a reliability required for medicinal purposes.

Most of the preparations manufactured or sold `for medicinal purposes, which contain insulin in solution at pH 3 or as a suspension with zinc or proteins, have not required insulin purified to the extent which is necessary to form stable solutions under substantially neutral conditions between pH 7 and 8. It is, for instance, assumed that insulin of a potency of 22 international units/ mg. is only pure, yet it is still frequently used and referred to in literature. However, in case of a purity higher than this, the potency is not sufficiently informative to indicate the particular type of insulin which could be suitable for the present purposes, as very small amounts of the mentioned undesirable impurities can upset the stability of the solution within the defined pH range.

It has now been observed that the absence of these undesirable impurities is associated with a very low content of protaminase in the insulin and with a capability of the insulin of forming solutions with a very limited optical density. In this respect, an increased optical density, caused either by light absorption or by light dispersion (scattering) because of turbidity, indicates the unsuitability of an insulin for the purposes of stable neutral solutions.

According to the present invention in one aspect therefore there is provided a stable and pharmaceutically acceptable solution of insulin, comprising beef insulin, which has a molecular weight of 57 34, substantially the following elementary analysis (percent): carbon 53.21, hydrogen, 6.63; nitrogen, 15.88; sulphur, 3.36 and oxygen 20.92 (by difference), and characteristically alanine, serine and valine in the 8th, 9th and 10th position of the A chain, and has (i) a protaminase content causing less than about 15% w./w. loss at 37 C. in 30 days when the insulin is converted into a protamine-containing isophane insulin suspension, (ii) an optical density (absorbence), corrected to a manifold diluted sample of less than about 0.5 as a 1% acid `solution in a 4 cm. cell at a wavelength of 396 my, (iii) an optical density (absorbence) of less than about 0.03 at a wavelength of 400 ma when measured 3 as a 40 international units/ml. solution in a 1 crn. cell at a pH of 7.4, after standing at 5 C. for eight days, with an instrument adapted to exclude scattered light, and (iv) an infra-red spectrum such as shown in the drawing; as an aqueous solution, adjusted to a pH between pH 7 and 8.

In another aspect there is provided a pharmaceutical preparation comprising beef insulin purified, as hereinbefore defined, in association with a therapeutically acceptable carrier sealed in a container as a sterile solution of a pH between pH 7 and 8. In a further aspect, methods are provided for producing a stable solution of beef insulin at a pH between pH 7 and 8 and a pharmaceutical preparation thereform according to the present invention.

The presence of protaminase may, for instance, be detected according to the method described in the British Pharmacopoeia 1963, page 407. This may conveniently be carried out by first converting a sample of the insulin with zinc, protamine and phenol into a suspension corresponding to the Isophane Insulin Injection of the Pharmacopoeia according to a method described in the specification of British Pat. No. 643,268 and by testing this preparation for protaminase. According to the method described in British Pat. 643,268 the precipitation of the protamine-insulin complex is carried out under isophanous conditions to achieve equilibrium conditions indicated by the same degree of opalescence in the supernatant liquor by the addition of either insulin or protamine.

The testing procedure consists of taking a small sample of this preparation, which consists of protamine insulin obtained under isophanous conditions, and dividing it into two equal poritions. The first portion is centrifuged, and the deposit washed with absolute alcohol, dried over phosphorus pentoxide at a pressure not exceeding 5 mm. Hg for 24 hours, and weighed. The second portion is maintained at 37 C. for 30 days, and subsequently centrifuged, the deposit being washed, dried and weighed, as above.

Beef insulin which fails to pass this test by showing a difference in weight of more than calculated on the first sample or an equivalent test for protaminase, is not suitable for forming stable and substantially neutral solution. It is preferred to use insulins showing a difference in weight of less than 10% in this test for the purposes of the present invention, although those with a difference between 10 and 15% may also be suitable.

In the method preferred for testing the optical density (absorbence) of the beef insulin in acid solution a 1% solution of insulin in 0.01 N hydrochloric acid is prepared and the solution is filtered through a Whatman (trademark) No. 42 grade filter paper sold by Messrs. W. & R. Baltson, Ltd., London. The optical density of the solution is measured in an appropriate 1, 2 or 4 cm. cell at 396 mp. against 0.01 N hydrochloric acid in a reference cell. A sample of the solution is diluted with 0.01 yN hydrochloric acid to a twentyfold volume, and the optical density of the diluted sample is again measured at 276.5 mp. against a reference sample. The following formula then gives a corrected optical density value F: (ea-0.253%) /percent concentration of the solution where ea is the optical density of the first solution at 396 mn, measured or calculated for a 4 cm. cell, eb is the optical density of the diluted solution at 276.5 mit measured in or calculated for a 1 cm. cell.

Beef insulin for which the corrected optical density value F is greater than 0.5 under these conditions is not suitable for forming a stable and substantially neutral solution. It is preferred to use insulins for which the F value is not greater than 0.3 (i.e. net transmittance of about 50%) for the purposes of the present invention, although those for which this is between 0.3 and 0.5 (i.e. net transmittance of about 31.5%) may also be suitable.

In the method preferred for testing the optical density of the beef insulin under substantially neutral conditions at pH 7.4, a solution (20 ml.) containing insulin (800 international units), sodium chloride mg), sodium acetate (28 mg), and methyl p-hydroxybenzoate (20 mg.) is prepared, adjusted to the above pH and stored at a temperature between 0 C. and 5 C. for 8 days. The optical density of the solution is then measured at 400 mp. in a 1 cm. cell by using a photometer adapted to exclude dispersed (scattered) light. A Unicam S.P. 500 spectrophotometer modified thus to measure turbidities have been found satisfactory for the purpose.

Beef insulin for which the optical density value is greater than 0.03 under these conditions is not suitable for forming a stable and substantially neutral solution. It is preferred to use insulins for which this optical density value is less than 0.025 for the purposes of the present invention, although those for which this is between 0.025 and 0.03 may also be suitable.

The optical density of a solution of insulin may be determined by any suitable method or instrument known in the art, and accordingly limits equivalent to the limit above can be calculated or determined.

The elementary analysis of beef insulin may be determined by means well known in the art, preferably by micro-analytical methods. Slight deviations from the theoretical values can always be expected, even in cases when insulin samples useful for solutions according to the present invention are analysed. These are due to small amounts of impurities or breakdown products, such. as desamino insulin and the like. Typical results for crystalline beef insulins are as follows (allowing for loss and ash):

Carbon Hydrogen Nitrogen Sulphur Loss Ash Theoretical. 53. 21 6. 63 15.88 3. 36

Sample No.'

ggg 0.1i s 8.75 1.70

Samples PA 3582, PA 3545 and PA 3596 showed satisfactorily low protaminase content and optical densities, as hereinbefore defined, and solutions prepared therefrom according to the invention remained clear, bright and pharmaceutically acceptable without any significant loss of biological activity after more than 3 months storage. Sample PA 3643 on the other hand, failed to comply with the purity requirements required by the present invention, and soon decomposed on standing when prepared as a solution for injection.

The infra-red spectrum of purified beef insulin exhibits characteristic absorption bands at the following frequencies 3065, 2963, 2938, 2877, 1663, 1520, 1455, 1395, 1240, 1176, 1128, 832 and 698 reciprocal c entimetres. It has been observed that the transmittance of beef insulin is increased on purification at 1395 cm1 relative to that at 1240 cm.1. Whilst the value at 1395 cm.n1 is slightly below that at 1240 crn.-1 with standard beef insulin the purification tends to reverse their relative position. A spectrum of purified beef insulin, suitable for the purposes of the present invention, is shown in the drawing.

Such purified beef insulins may be obtained by the use of purification methods eficient enough to remove the undseirable impurities. Depending on the quality and the type of pancreas from which the insulin is produced, various methods may be employed, which are well known in the field of natural polypeptide and protein chemistry and technology is general, and in the processing of insulin in particular. However, sometimes only a combination of these methods can provide the type of insulin required for the present purposes.

These methods of purification may, for example, nclude the removal of some other polypeptides and impurities while they are insoluble at their isoelectric points, as described, for instance, by M. Somogyi et al., I. Biol. Chem., 1924, 60, 31-48, which separates non-insulin proteins at about pH4 and pH6 to 8. Other methods may use repeated recrystallisations from a citrate buffer in the presence of zinc, countercurrent fractionation in a butanol/ aqueous system, adsorption or ionexchange chromatography or a precipitation with picric acid.

It is known that apart from impurities, the stability of an insulin solution under substantially neutral conditions depends on the pH and temperature, and also on the concentrations of crystallisation promoting metals. Beef insulin having the purity according to the requirements of the present invention has been found to tolerate more zinc in the absence of chelating compounds than it has originally been assumed. Bright and stable solutions have been obtained in the whole range between pH 7 and pH 8 with beef insulins containing up to 0.6% zinc, and above pH 7.3 with those containing 0.8% zinc.

The risk of crystallisation can be reduced by adjusting the solution to a higher pH value within the range or keeping it at room temperature rather than at a lower temperature, and the eliect of metals can be impeded by the addition of a chelating agent, or by the use of a buffering agent which has chelating properties. Thus in the presence of a phosphate or citrate buffer, which have slight chelating properties, a higher concentration of a crystallisation promoting metal is tolerated in the solution than otherwise. In practice, a crystalline or insulin of the required purity, containing the usual 0.3% to 0.4% zinc, may conveniently be used Without the necessity of adding agents having chelating properties.

[For the purpose of pharmaceutical preparations according to the present invention, the solution is preferred to contain 20, 40 or 80 international units per millilitre of puried beef insulin as hereinbefore defined, and may also contain buiering agents and salt to provide isotonic conditions. As buffering agents, pharmaceutically acceptable acetates or borosuccinates may be used, but other buffers, known in the art for this purpose and containing for instance maleate, citrate or phosphate ions, may also be employed. Usually a preservative, such as methyl or proply p-hydroxybenzoate is also added to the solution.

After the solution has been formed, it can be sterilised for instance by filtration through a procelain filter candle, and can then be aseptically distributed into ampoules or vials and sealed. These may conveniently contain to 10 ml. insulin solution representing a total of 200 to 400 international units per ampoule or vial.

The solution in bulk or the pharmaceutical preparations containing a predetermined dosage of insulin according to the present invention, are suitable for being stored for 'a prolonged period even at 5 C., Without showing signiiicant signs of decomposition, deterioration, deposition or loss of biological activity. It has been shown that these preparations can be admixed with neutral prolonged acting preparations, such as Isophan Insulin Injection, Protamine Zinc Insulin Injection and Insulin Zinc Suspensions, and the mixture shows both quick acting and prolonged acting properties when administered. They cause less damage to subcutaneous tissue around the injection site than does the acid Insulin Injection.

The following examples illustrate the invention.

EXAMPLE 1 A sample of crystalline ox insulin (9.3 g.), which has been purified by removing insoluble polypeptides at a pH about 4 and between pH 6 and 8, having a potency of 24.7 international units/mg., a zinc content of 0.38%, an F value of 0.176, and a a satisfactory test for protaminase (ditference below 10%) and for optical density at pH 7.4 (below 0.020), was dissolved in 0.1 N hydrochloric acid (120 ml.) and the solution was diluted with pyrogen free distilled water to approx. 1.36 litre. 0.1 N sodium hydroxide (240 ml.) was slowly added under shaking and the solution was diluted to 1.8 litre.

'An acetate solution (5 litre), which had been made up by dissolving sodium acetate (10.0 g.), sodium chloride (50 g.) and methyl p-hydroxybenzoate (7.14 g.) in hot pyrogen free distilled and adjusting the volume to 5 litres after cooling, was added to the solution containing the insulin.

The resulting soluton had a pH of 7.4, and was subsequently sterilized =by ltration through a procelain filter candle and aseptically distributed into 10 ml. ampoules and vials, and sealed. The sterile product had a concentration of 40 international units/ml.

A sample of the sealed ampoules and vials was subjected to standard testing and was found satisfactory. Another sample was stored at 50 C. and a further sample at 25 C. After three months of storage, these were clear and bright without a significant loss of biological activity, and were found satisfactory for use. After 15 months storage the samples were still acceptable in these respects.

EXAMPLE 2 Purified crystalline ox insulin (4000 international units), having a potency of 23.3 international units/mg. a zinc content of 0.37%, an F value of 0.197, and a satisfactory test for protaminase (difference below 10% and for optical density (below 0.025) was dissolved in 0.1 N hydrochloric acid (2.0 ml.) and the solution was diluted with pyrogen free distilled water to approx. 25 ml. of 0.1 N sodium hyroxide (4.0 ml.) was slowly added under shaking 'and the solution was diluted to 30 ml. A borosuccinate solution, which had been made up by dissolving borax (0.95 g.), succinic acid (0.295 g.), sodium chloride (0.70 g.), and methyl p-hydroxybenzoate (0.10 g.) in hot pyrogen free distilled water and adjusting the volume to 70 ml. after cooling, was added to the solution containing the insulin.

The mixture 'had a pH of 7.4, and was sterilized and aseptically distributed into 5 ml. of ampoules. Satisfactory results were obtained on storing the solution, as in Example l.

EXAMPLE 3 A sample of crystalline ox insulin, which was used in Example l (1,600,000 units), was dissolved in 0.1 N hydrochloric Iacid (600 ml.) and the solution was diluted with pyrogen free distilled water to 4 liters. 0.1 N sodium hydroxide (1,200 ml.) was slowly added under constant mixing, while the insulin precipitated and redissolved. The solution was then diluted to 6000 ml. with pyrogen free distilled water.

'An acetate solution, which had been made up by dissolving sodium acetate (30 g.), sodium chloride (150 g.) and methyl p-hydroxybenzoate (21.42 g.) in hot pyrogen free distilled water and adjusting the volume to 15,000 ml., was slowly added to the solution containing the insulin with constant mixing until 20,000 ml. buffered solution was obtained.

The solution was sterilized by filtration through a procelain rfilter candle and aseptically distributed into l0 ml. vials, and the lvials were sealed. The sterile product had a concentration of international units/ml., and was stable after three months storage and pharmaceutically acceptable.

EXAMPLE 4 Citrate buffer, suitable for use in this and subsequent examples was prepared by dissolving citric acid (3.6 kg.) in distilled water (72 1.), and adjusting the pH to pH 5.7 to 5.8 with sodium hydroxide. To this solution acetone (20.4 l.) was added with stirring.

Crystalline ox insulin (4 g.), from a better than average production batch crystallised from a citrate buffer, was dissolved in water (100 ml.) at pH 3, containing 1% zinc chloride solution (2 ml.). An aqueous solution (100 ml.), containing sodium acetate (2.72 g.), sodium chloride (14 g.) and N sodium hydroxide solution (4 ml.), was added. The amorphous suspension was stirred for 48 hours at pH 5.4 to 5.5 at room temperature. After cooling to a temperature below C., acetone at a temperature below 10 C. (100 ml.) was added with stirring, and the mixture was left to stand for one hour. The precipitate was collected, dissolved in water (160 ml.) adjusted to pH 3 with citrate, and the pH of the solution was adjusted to 6.6. A citrate buffer having a volume 5/6 of the adjusted solution, was added slowly, and the precipitated crystals were collected and dried with acetone and ether. The purified insulin `had an optical density at pH 7.4 of 0.011 unit.

EXAMPLE 5 Ox insulin from a normal batch, which had been crystallised from citrate buffer, was dissolved in water to give a 2% w./w. solution. The pH of the solution was adjusted to pH 6.6, and 1% zinc chloride solution was added (40 ml./1.). The mixture was stirred and 5/6 volume of citrate buffer was slowly added. The precipitated crystals were collected, redissolved in water and the crystallisation procedure was repeated four times.

The purified insulin had an F value below 0.5 and a satisfactory test for protaminase and optical density at pH 7.4 (0.015 unit). It gave a satisfactory and stable solution when this was prepared according to the method given in Example 1.

EXAMPLE 6 Ox insulin from a normal production batch, which had been crystallised from citrate buffer, was dissolved in water containing p-toluene sulphonic acid to give a 1% insulin solution (150 ml.).

The solution was filtered under pressure through a column, 2 cm. long and 1 cm. diameter, of tightly packed cellulose fibres derived from pulped Whatman No. 1 filter paper. The effluent solution was adjusted to the isoelectric point, the precipitate was collected and dissolved in water. After adjusting the pH to 6.6, 1% zinc chloride solution (40 ml./1.) was added and 5/6 volume of citrate buffer was slowly added. The precipitated crystals were collected and dried.

The purified insulin had an F value below 0.5 and a satisfactory test for protaminase and optical density at pH 7.4 (0.016 unit). It gave a satisfactory and stable solution when this was prepared according to the method given in Example 1.

EXAMPLE 7 Crystallised ox insulin (5 g.) from a normal production batch, which had been crystallised from citrate buffer, was distributed in the four initial tubes of a 120 tube Craig apparatus suitable for counter-current stage-wise liquid-liquid extractions. The volume of each aqueous phase in each tube was 40 ml. containing 1% dichloroacetic acid. The same volume of sec-butanol was used as the organic phase. At each transfer shaking for one minute was followed by fifteen minutes settling, and at 120 transfers the settling time was reduced to three minutes.

After a total of 710 transfers a main fraction and a faster running fraction were isolated. Each of these fractions was converted to the hydrochloride after a precipitation with picrate, to give a main fraction (3.15 g.) and a faster running fraction (0.883 g.). Part of the main fraction was recrystallised from citrate buffer and the optical density values at pH 7.4 of the various groups were:

Units Main fraction hydrochloride 0.026 Main fraction hydrochloride recrystallised 0.01 Fast running fraction hydrochloride 0.245

The purified, recrystallised main fraction had an F value below 0.5 and a satisfactory test for protaminase. It gave a satisfactory and stable solution when this was prepared according to the method given in Example 1.

I claim:

1. A stable and pharmaceutically acceptable solution of insulin, comprising (a) beef insulin which has a molecular weight of 5734, substantially the following elementary analysis (percent): carbon 53.21, hydrogen 6.63, nitrogen 15.88, sulphur 3.36, and oxygen 20.92, and characteristically alanine, serine, and valine in the 8th, 9th and 10th position of the A chain and alanine in the 30th position of the B chain, and has (1) a protaminase content causing less than about 15% w./w. loss at 37 C. in 30 days when the insulin is converted into a protamine-containing isophane insulin suspension,

(2) an optical density, corrected to a manifold diluted sample, of less than about 0.5 as a 1% acid solution in a 4 cm. cell at a wavelength of 396 ma.

(3) an optical density of less than about 0.03 at a wavelength of 400 mit when measured as a 40 international units/ml. solution in a 1 cm. cell at a pH of 7.4, after standing at 5 C. for eight days, with an instrument adapted to exclude scattered light, and

(4) an infra-red spectrum such as shown in the drawing.

(b) said beef insulin, in an aqueous solution, adjusted to a pH between pH 7 and pH 8, and

(c) said solution containing from about 20 to about international units/ ml. of said beef insulin.

2. A stable and pharmaceutically acceptable solution of insulin according to claim 1, which contains between 0.3% and 0.4% zinc in proportion to insulin.

3. A stable, pharmaceutically acceptable solution of insulin according to claim 1, in the form of an ampoule, and wherein said solution is sterile.

References Cited OTHER REFERENCES 2,595,278 5/1952 Maxwell et al 260112.7 3,364,116 1/1968 Bodanszky 424-178 FOREIGN PATENTS 840,870 7/1960 Great Britain 260-1l2.7

JEROME D. GOLDBERG, Primary Examiner V. D. TURNER, Assistant Examiner 

